Protein-protein interactions are an essential key in all biological processes, from the replication and expression of genes, to the morphogenesis of organisms. Protein-protein interactions govern, amongst others, ligand-receptor interaction and the subsequent signaling pathway; they are important in assembly of enzyme subunits, in the formation of biological supramolecular structures such as ribosomes, filaments and virus particles, and in antigen-antibody interactions.
Researchers have developed several approaches in attempts to identify protein-protein interactions. A major breakthrough was obtained by the introduction of the genetic approaches, of which the yeast two-hybrid (Fields and Song, 1989) is the most important one. Although this technique became widely used, it has several drawbacks. The fusion proteins need to be translocated to the nucleus, which is not always evident. Proteins with intrinsic transcription activation properties may cause false positives. Moreover, interactions that are dependent upon secondary modifications of the protein such as phosphorylation cannot be easily detected.
Several alternative systems have been developed to solve one or more of these problems.
Approaches based on phage display do avoid nuclear translocation. WO9002809 describes how a binding protein can be displayed on the surface of a genetic package, such as a filamentous phage, wherein the gene encoding the binding protein is packaged inside the phage. Phages that bear the binding protein that recognizes the target molecule are isolated and amplified. Several improvements of the phage display approach have been proposed, as described, e.g., in WO9220791, WO9710330, and WO9732017.
However, all these methods suffer from the difficulties that are inherent at the phage display methodology: the proteins need to be exposed at the phage surface and are so exposed to an environment that is not physiologically relevant for the in vivo interaction. Moreover, when screening a phage library, there will be a competition between the phages that results in a selection of the high-affinity binders.
U.S. Pat. No. 5,637,463 describes an improvement of the yeast two-hybrid system, whereby it can be screened for modification-dependent protein-protein interactions. However, this method relies on the co-expression of the modifying enzyme, which will exert its activity in the cytoplasm and may modify enzymes other than the one involved in the protein-protein interaction, which may, on its turn, affect the viability of the host organism.
An interesting evolution is described in U.S. Pat. No. 5,776,689, by the so-called protein recruitment system. Protein-protein interactions are detected by recruitment of a guanine nucleotide exchange factor (Sos) to the plasma membrane, where Sos activates a Ras reporter molecule. This results in the survival of the cell that otherwise would not survive in the culture conditions used. Although this method has certainly the advantage that the protein-protein interaction takes place under physiological conditions in the submembrane space, it has several drawbacks. Modification-dependent interactions cannot be detected. Moreover, the method is using the pleiotropic Ras pathway, which may cause technical complications, such as the occurrence of false positives.
A major improvement in the detection of protein-protein interactions was disclosed in WO0190188, describing the so-called Mappit system. The method, based on a cytokine receptor, not only allows a reliable detection of protein-protein interactions in mammalian cells, but also modification-dependent protein interactions can be detected, as well as complex three-hybrid protein-protein interactions mediated by a small compound (Caligiuri et al., 2006). However, although very useful, the system is limited in sensitivity and some weak interactions cannot be detected. Moreover, as this is a membrane-based system, nuclear interactions are normally not detected. Recently, a cytoplasmic interaction trap has been described, solving several of those shortcomings. However, all of these “genetic” systems rely on the overexpression of both interaction partners, which may result in false positives due to the artificial increase in concentration of one or both of the interaction partners.
As an alternative for the genetic protein-protein interaction detection methods described above, biochemical or co-purification strategies, combined with mass spectrometry-based proteomics (Paul et al., 2011; Gingras et al., 2007), can be used. For the co-purification strategies, a cell homogenate is typically prepared by a detergent-based lysis protocol, followed by capture using a (dual) tag approach (Gavin et al., 2002) or via specific antibodies (Malovannaya et al.). The protein complex extracted from the “soup” of cell constituents is then expected to survive several washing steps, mostly to compensate for the sensitivity of contemporary MS instruments, before the actual analysis occurs. There are no clear guidelines on the extent of washing or on available buffers and their stringency. Most lysis and washing protocols are purely empirical in nature and were optimized using model interactions. It is, therefore, hard to speculate on the loss of factors during these steps (false negatives), or the possibility of false interactions due to loss of cellular integrity (false positives). Use of metabolic labeling strategies allows separation between the proteins sticking to the purification matrix, and between the proteins that associate specifically to the bait protein. Depending on the purification conditions and the sensitivity of the MS instruments used, it is no exception to find more than 1000 proteins in the eluted fraction of a gel-free AP-MS experiment.
There is a further need for co-purification techniques, isolating the protein complexes in their physiological environment, but wherein the complex is protected during the further purification and analysis.
The evolutionary stress on viruses promotes highly condensed coding of information and maximal functionality for small genomes. Accordingly, for HIV-1, it is sufficient to express a single viral protein, the p55 GAG protein, to allow the efficient production of virus-like particles (VLPs) from cells (Gheysen et al., 1989; Shioda and Shibuta, 1990). The p55 GAG protein consists of different parts, which are processed by HIV protease upon maturation of the particle into a functional infectious particle. The N-terminal matrix protein part ensures binding to the membrane via myristoylation and ensures budding (Bryant and Ratner, 1990). The Capsid protein forms the cone-shaped viral core after processing, while the nucleocapsid protein and the p6 protein bind to and protect the viral RNA. The p55 GAG protein is highly mobile before accumulation in cholesterol-rich regions of the membrane, where multimerization actually initiates the budding process (Gomez and Hope, 2006). A total of 4000-5000 GAG molecules are required to form a single particle with a size of about 145 nm (Briggs et al., 2004).